The present invention relates to a novel antibacterial protein and compositions, in the form of pharmaceutical compositions, human food compositions, and animal feedstuffs comprising said protein to be used in the therapeutic and/or prophylactic treatment of infections caused by bacteria, in particular Streptococcus pneumoniae and/or Haemophilus influenzae, as well as a method for diagnosing infections caused by said bacteria.
The object of the present invention is to obtain a protein and compositions containing said protein for prophylactic and/or therapeutic treatment of infections caused by bacteria, in particular Streptococcus pneumoniae and Haemophilus influenzae in the upper airways, ear-nose-and-throat infections, but also in the lower airways, e.g., the lungs by preventing adhesion of and/or causing a bactericidal effect on these bacteria. A further object is to be able to diagnose infections caused by these bacteria.
Natural antimicrobial compounds exist in secreted form as well as in cells of immune and non-immune origin.
Human milk has been used as a source for the purification of such compounds. These previously known compounds include specific antibodies to the micro-organism surface structure, casein, lysozyme, and oligosaccharides. The mechanism of action differs between the groups of antimicrobial molecules. Antibodies and receptor analogues prevent micro-organism adherence to mucosal surfaces. Lysozyme attacks the cell wall etc.
The term bacterial adherence denotes the binding of bacteria to mucosal surfaces. This mechanic association is a means for the organism to resist elimination by the body fluids, and to establish a population at the site where relevant receptors are expressed. In most cases where the mechanisms of attachment have been identified it is a specific process. The bacterial ligands, commonly called adhesins bind to host receptors. For Gram-negative bacteria, the adhesins are commonly associated with pili or fimbriae, rigid surface organelles that help bacteria to reach the appropriate receptor in the complex cell surface. The fimbriae function as lectins, i.e. they show specificity for receptor epitopes provided by the oligosaccharide sequences in host glyco-conjugates (13). For Gram-positive bacteria, on the other hand, the adhesins are not expressed as a surface organell, but rather linked to cell wall components and lipoteichoic acids (21,22). The receptor epitopes for Gram positive bacteria may consist of oligosaccharide sequences but can also be provided by peptides e.g. in connective tissue proteins (10).
The functional consequences of adherence depend on the virulence of the bacterial strain, and on the form of the receptor. When cell-associated, the ligand receptor interaction facilitates colonization and tissue attack (8). When secreted the receptor molecule will occupy the adhesins, and competitively inhibit attachment to the corresponding cell-bound receptor. Human milk is a rich source of such competing soluble receptor molecules.
The ability of specific antibodies to inhibit attachment is well established. This was first demonstrated for Vibrio cholera and oral streptococci. The anti-adhesive antibodies may act in either of two ways:
1) Antibodies to the receptor binding sites of the adhesin competitively inhibit receptor interaction or
2) antibodies to bacterial surface molecules which are not directly involved in adherence may agglutinate the bacteria and thereby reduce the number of organisms available for binding.
In either of the above cases the anti-adhesive activity of the antibody is attributed to the specificity of the antigen-combining site. Recently an alternative mechanism of interaction between secretory IgA and E. coli based on lectin-carbohydrate interactions was identified.
Human milk drastically inhibits the attachment of Streptococcus pneumoniae and Haemophilus influenzae to human nasopharyngeal epithelial cells. It contains antibodies to numerous surface antigens on these organisms, e.g., the phosphoryl choline and capsular polysaccharides of S. pneumoniae, the lipopolysaccharide and outer membrane proteins of H. influenzae. Accordingly, some of the anti-adhesive activity in milk resides in the immunoglobulin fraction.
The remaining anti-adhesive activity in the non-immunoglobulin fraction of milk may be explained by two types of molecules: free oligosaccharides and glycoproteins in the casein fraction.
Human milk is unique with regard to its content of complex carbohydrates. The free oligosaccharide fraction of milk is dominated by the lactoseries and with improving methods of isolation and characterization of carbohydrates more than 130 oligosaccharides containing up to 20 monosaccharides per molecule have been identified.
An anti-adhesive activity against S. pneumoniae in a low molecular weight fraction ( less than 5 kDa) of milk was explained by the free oligosaccharides. In contrast there was no such effect against H. influenzae (15).
An anti-adhesive activity of high molecular weight components of milk was localized to the casein fraction. Human casein drastically reduced the adherence both of S. pneumoniae and H. influenzae (15). This effect was species specific.
Alpha-lactalbumin is a mettaloprotein, which shows some degree of heterogeneity depending on Ca(II) saturation and/or glycosylation (1). Alpha-lactalbumin acts as a specifier protein in the lactose synthase system. During lactation, alpha-lactalbumin is formed in the mammary gland and it alters the substrate specificity of the galactosyltransferase enzyme from N-acetyl glucosamine (GlcNAc) to glucose (Glc), enabling lactose synthesis to take place: 
Multiple forms of bovine, pig, sheep and goat alpha-lactalbumin have been isolated and well characterized (2, 3). These multiple forms differ in a few amino residues or the number of disulphide bonds (4, 5) but are all active in the lactose synthase system. The physiological relevance or functions of these different forms of alpha-lactalbumin are not known. Alpha-lactalbumin has undergone a high rate of evulotionary change and it shows homology with lysozyme (1). These two proteins are thought to originate from the same ancestral protein. Whereas lysozyme is known as an anti-bacterial agent, alpha-lactalbumin has not yet been found to have anti-bacterial functions.
The present invention describes the identification of a new anti-bacterial protein or group of proteins from milk. The protein comprises a multimeric form of alpha-lactalbumin.
In the following this protein, or group of proteins, is abbreviated ALLP, Anti-adhesive Lactalbumin Like Protein.
The term antimicrobial or anti-bacterial protein used in the context of the present invention means here and in the following a protein which inhibits adherence of micro-organisms to tissue and/or exerts a bactericidal effect on microorganisms.
Further characteristics of the invention will be evident from the accompanying claims.